During this reporting period the Laboratory of Genetics and Physiology has made major progress in understanding of strengths and weaknesses of CRISPR/Cas9-based genome editing technologies. These tools are widely used for the introduction of mutations into the mammalian genome, both for basic and translational research. We have made advances in understanding molecular mechanisms on how hormones activate genes in the mammary gland. Fidelity of CRISPR/Cas9 genome editing in the mouse genome CRISPR/Cas9 genome editing technologies provide unprecedented opportunities to conduct tailored engineering of the genome. It permits scientists to address fundamental problems, both in basic science and in translational medicine. However, successful use of CRISPR/Cas9 editing requires exceptional fidelity, i.e. only the introduction of intended genetic changes and a complete absence of unintended changes. This is particularly important for its application in gene therapy where precision cannot be compromised. Unwanted genetic changes introduced by CRISPR/Cas9 could result in the disruption of vital cellular functions. It was therefore critical to determine the degree of fidelity of CRISPR/Cas9 genome editing. Previously we had discovered that CRISPR/Cas9 editing in the mammalian (mouse) genome results in a high frequency of large deletions and insertions at target sites (PMID: 28561021). A key concern of CRISPR/Cas9 genome editing is the potential for creating mutations at non-target sites and the identification of hundreds of non-targeted mutations in CRISPR/Cas9-treated mice had been reported by others (PMID: 28557981). Shortcomings of that analysis were the failure to compare parents to progeny, a necessary prerequisite for discriminating de novo mutations from pre-existing variants in the strain background and the small number of samples examined (one control plus two CRISPR-Cas9-edited animals). As discussed in an Editorial in Nature Methods, there is a need for an understanding of in vivo genomic effects of CRISPR. In this reporting period we have addressed this question, designed a parent-progeny study and conducted unbiased whole genome sequencing (WGS) on six CRISPR-Cas9-edited mice, six control mice and their 24 wild-type parents (C57BL6/N strain) with the goal of determining the frequency of de novo mutations in any of the more than three billion bases of the mammalian genome. We did not detect an increased number of spurious off-target mutations in edited mice (PMID: 30275594). However, unwanted deletions and insertions at target sites remain a concern (PMID: 28557981). Evaluation of deaminase base editing in manipulating the genome Since CRISPR/Cas9 genome editing causes unwanted genetic changes in the mammalian genome with potentially adverse consequences, we decided to evaluate a new and advanced editing technology. Base editing directly converts a target base pair into a different base pair in the genome of living cells without introducing double-stranded DNA breaks. While cytosine base editors (CBE) and adenine base editors (ABE) are used to install and correct point mutations in a wide range of organisms, the extent and distribution of off-target edits in mammalian embryos have not been studied in detail. We analyzed on-target and proximal off-target editing at 13 loci by a variety of CBEs and ABE in more than 430 alleles generated from mouse zygotic injections using newly generated and published sequencing data (PMID: 30442934). ABE predominantly generates anticipated AT-to-GC edits. Among CBEs, SaBE3 and BE4, result in the highest frequencies of anticipated CG-to-TA products relative to editing byproducts. Together, these findings highlight the remarkable fidelity of ABE in mouse embryos and identify preferred CBE variants when fidelity in vivo is critical. A particular challenge in genome engineering has been the simultaneous introduction of mutations into linked (located on the same chromosome) loci. Although CRISPR/Cas9 has been widely used to mutate individual sites, its application in simultaneously targeting of linked loci is limited as multiple nearby double-stranded DNA breaks created by Cas9 routinely result in the deletion of sequences between the cleavage sites (PMID: 28561021). Base editing is a newer form of genome editing that directly converts CG-to-TA, or AT-to-GC, base pairs without introducing double-stranded breaks, thus opening the possibility to generate linked mutations without disrupting the entire locus. In this reporting period we addressed this issue using deaminase base editors (PMID: 30733567). Through the co-injection of two base editors and two sgRNAs into mouse zygotes, we introduced CG-to-TA transitions into two cytokine-sensing transcription factor binding sites separated by 9kb. We determined that one enhancer activates the two flanking genes in mammary tissue during pregnancy and lactation. The ability to introduce linked mutations simultaneously in one step into the mammalian germline has implications for a wide range of applications, including the functional analysis of linked cis-elements creating disease models and correcting pathogenic mutations. Structure and function of super-enhancers Super-enhancers comprise dense transcription factor platforms highly enriched for active chromatin marks. A paucity of functional data led us to investigate over the past two years the role of super-enhancers in the mammary gland, an organ characterized by exceptional gene regulatory dynamics during pregnancy. Previously we have identified 440 mammary-specific super-enhancers where the master regulator STAT5A integrates the glucocorticoid receptor, H3K27ac and MED1 to highly activate genetic programs during pregnancy (PMID: 27376239). As part of this study we discovered a hierarchy of enhancers within super-enhancers, pointing to complex interactions between regulatory elements. In the current reporting period, we explored how super-enhancers control genetic programs throughout lactation (PMID: 30285185). The mammary luminal lineage relies on the common cytokine-sensing transcription factor STAT5 to establish super-enhancers during pregnancy and initiate a genetic program that activates milk production. As pups grow, the greatly increasing demand for milk requires progressive differentiation of mammary cells with advancing lactation. Here we investigate how persistent hormonal exposure during lactation shapes an evolving enhancer landscape and impacts the biology of mammary cells. Employing ChIP-seq, we uncover a changing transcription factor occupancy at mammary enhancers, suggesting that their activities evolve with advancing differentiation. Using mouse genetics, we demonstrate that the functions of individual enhancers within the Wap super-enhancer evolve as lactation progresses. Most profoundly, a seed enhancer, which is mandatory for the activation of the Wap super-enhancer during pregnancy, is not required during lactation, suggesting compensatory flexibility. Combinatorial deletions of structurally equivalent constituent enhancers demonstrated differentiation-specific compensatory activities during lactation. We also demonstrate that the Wap super-enhancer, which is built on STAT5 and other common transcription factors, retains its exquisite mammary specificity when placed into globally permissive chromatin, suggesting a limited role of chromatin in controlling cell specificity. Our studies unveil a previously unrecognized progressive enhancer landscape where structurally equivalent components serve unique and differentiation-specific functions.